Mutational Analysis of TGF beta Function. Congenital heart disease occurs in nearly 1 percent of all live births in the United States. Over 1/3 of these involve ventral and atrial septal defects and outflow tract defects. About a quarter of all heart defects result in critical disease, and even with modern medical practices about 1/3 of these still do not support life past one year. Even though congenital heart defects have both environmental as well as genetic components, genetic models of these abnormalities will be useful for identifying the molecular pathways through which both environmental and genetic factors can interfere. Therefore, identification of these pathways will likely lead to improved diagnostic, intervention and treatment strategies that will be applicable to both the environmental and genetic components of congenital heart disease. Transforming Growth Factor beta 2 (TGFbeta2) knockout mice have congenital heart abnormalities that are commonly found in humans: double outlet right ventricle, double inlet left ventricle, ventral and atrial septal defects, and valve defects. For the 4-chambered heart to form with proper alignment of the great vessels with their respective ventricles, considerable morphogenesis and remodeling must occur. Defects in either cushion formation or the subsequent myocardialization of those cushions could lead to such congenital abnormalities. We propose an in depth analysis of 1) the process of cushion formation, in which an epithelial- mesenchymal transformation of the endocardial cells into cushion mesenchyme occurs, and 2) the process of myocardialization, in which inner curvature myocardial cells, with the help of neural crest cells, invade and muscularize the cushion mesenchyme. We will apply immunohistochemical, in situ hybridization and electron mircroscopy techniques to normal and knockout embryonic hearts to analyze alterations in expression of the cell adhesion, migration, extracellular matrix and differentiation molecules that may be involved in these processes. We will also determine which cells are responsible for producing the TGFbeta2 needed for these processes to occur normally by transgenic rescue and conditional knockout, both in myocardial and neural crest cells. Finally, we will use functional genomics to discover new candidate genes involved in these essential processes of heart development. With this study we will obtain a much better understanding of the molecular mechanisms underlying the most common congenital heart abnormalities in man.